Drug resistance of pathogenic microbes in healthcare and community settings is a quickly growing and extremely dangerous health threat. There is consequently a critical need for the development of alternative therapeutics to tackle drug resistance. The objective of this R01 application is to investigate a novel non-antibiotic approach, antimicrobial blue light (aBL), to target/treat microbes including drug-resistant strains In this application, we propose five specific aims. In Aim 1, we will determine the susceptibility f a wide range of important microbes to aBL at 405, 415 or 470 nm wavelength. Both planktonic cultures and biofilms will be studied. In Aim 2, we will determine whether microbes can develop resistance to aBL; and elucidate the molecular mechanism of mutant development if aBL resistance develops. In Aim 3, we will investigate the potential side effects of aBL on relevant host cells. In Aim 4, we will elucidate the mechanism of action of aBL. The study will be carried out in vitro by looking for a correlation between the level of endogenous photosensitizers in microbial cells and corresponding aBL susceptibility of microbes. Isogenic strains of representative microbes that differ only in the level of endogenous photosensitizers will be used. In Aim 5, we will demonstrate the effectiveness of aBL for prophylaxis and treatment of representative infections (urinary tract infections, surgical site infections, etc.) in murine modes. Both monomicrobial and polymicrobial infections will be studied. aBL will be initiated at 30 min, 4 h or 48 h after microbial inoculation. Bioluminescence imaging will be performed to noninvasively monitor in real time the extent of infection in living animals. The efficacy found with aBL will be compared with that of clinically used antibiotics. Successful completion of these specific aims will provide the foundation required to assess the effectiveness (Aims 1 and 5), potential side effects (Aims 2 and 4), and mechanism of action (Aim 3) of aBL inactivation of microbes, and will help establish protocols for the use of this therapeutic approach.